title: A label-free LC/MS/MS-based enzymatic activity assay for the detection of genuine caspase inhibitors and SAR development.
abstract: The resurgence of interest in caspases (Csp) as therapeutic targets for the treatment of neurodegenerative diseases prompted us to examine the suitability of published nonpeptidic Csp-3 and Csp-6 inhibitors for our medicinal chemistry programs. To support this effort, fluorescence-based Csp-2, Csp-3, and Csp-6 enzymatic assays were optimized for robustness against apparent enzyme inhibition caused by redox-cycling or aggregating compounds. The data obtained under these improved conditions challenge the validity of previously published data on Csp-3 and Csp-6 inhibitors for all but one series, namely, the isatins. Furthermore, in this series, it was observed that the nature of the rhodamine-labeled substrate, typically used to measure caspase activity, interfered with the pharmacological sensitivity of the Csp-2 assay. As a result, a liquid chromatography/tandem mass spectrometry-based assay that eliminates label-dependent assay interference was developed for Csp-2 and Csp-3. In these label-free assays, the activity values of the Csp-2 and Csp-3 reference inhibitors were in agreement with those obtained with the fluorogenic substrates. However, isatin 10a was 50-fold less potent in the label-free Csp-2 assay compared with the rhodamine-based fluorescence format, thus proving the need for an orthogonal readout to validate inhibitors in this class of targets highly susceptible to artifactual inhibition.
authors.affiliation: CHDI Management, Inc., Los Angeles, CA 90045, USA. michel.maillard@chdifoundation.org
authors.forename: Michel C
authors.initials: MC
authors.lastname: Maillard
authors.forename: Celia
authors.initials: C
authors.lastname: Dominguez
authors.forename: Mark J
authors.initials: MJ
authors.lastname: Gemkow
authors.forename: Florian
authors.initials: F
authors.lastname: Krieger
authors.forename: Hyunsun
authors.initials: H
authors.lastname: Park
authors.forename: Sabine
authors.initials: S
authors.lastname: Schaertl
authors.forename: Dirk
authors.initials: D
authors.lastname: Winkler
authors.forename: Ignacio
authors.initials: I
authors.lastname: Muñoz-Sanjuán
mesh_terms: D053143:Caspase 2 / Q000378:metabolism; D053148:Caspase 3 / Q000378:metabolism; D053178:Caspase 6 / Q000378:metabolism; D061945:Caspase Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D002853:Chromatography, Liquid / Q000379:methods; D015195:Drug Design; D057075:Enzyme Assays / Q000379:methods*; D007510:Isatin / Q000737:chemistry / Q000494:pharmacology; D019636:Neurodegenerative Diseases / Q000378:metabolism; D009842:Oligopeptides / Q000494:pharmacology; D053719:Tandem Mass Spectrometry / Q000379:methods
chemical_list: D061945:Caspase Inhibitors; D009842:Oligopeptides; C099158:acetyl-aspartyl-glutamyl-valyl-aspartal; C110923:acetyl-valyl-isoleucyl-aspartyl-aldehyde; D007510:Isatin; D053143:Caspase 2; D053148:Caspase 3; D053178:Caspase 6
keywords: LC/MS/MS assay; artifactual inhibition; caspases; nonpeptidic inhibitors